Polymer-Encapsulated Viral Vectors for Genetic Therapy

Abstract

The present technology relates to gene delivery vehicles comprising a retroviral vector coated with a polymer or a mixture of polymers to form a nanoparticle. The retroviral vectors comprise a transgene and in certain embodiments lack envelope protein. The technology includes a method of making the gene delivery vehicles and a method of treating a disease by administering the gene delivery vehicles.

What is claimed is: 
     
         1 . A nanoparticle comprising a viral vector coated with a polymer or a mixture of polymers, wherein the viral vector comprises a transgene, and wherein the nanoparticle functions as a vehicle for delivering the transgene to eukaryotic cells. 
     
     
         2 . The nanoparticle of  claim 1 , wherein the viral vector lacks envelope protein. 
     
     
         3 . The nanoparticle of  claim 1  or  claim 2 , wherein the polymer or the mixture of polymers comprises a poly(beta-amino ester) having the formula 
       
         
           
           
               
               
           
         
         wherein each Pep is an oligopeptide; wherein R is OH, CH 3 , or cholesterol; and wherein m ranges from 1 to 20. n ranges from 1 to 100, and o ranges from 1 to 10. 
       
     
     
         4 . The nanoparticle of  claim 3 , wherein each oligopeptide comprises at least three amino acid residues selected from the group consisting of arginine (R), glutamic acid (E), lysine (K), aspartic acid (D), histidine (H), and cysteine (C). 
     
     
         5 . The nanoparticle of  claim 3  or  claim 4 , wherein the amino acid sequence of at least one of the oligopeptides is CRRR (SEQ ID NO:1), or CHHH (SEQ ID NO:2), or CKKK (SEQ ID NO:3), or CEEE (SEQ ID NO:4), or CDDD (SEQ ID NO:5). 
     
     
         6 . The nanoparticle of any of the preceding claims, wherein the polymer or mixture of polymers has a net positive charge or a net negative charge at pH 7. 
     
     
         7 . The nanoparticle of any of the preceding claims, wherein the nanoparticle contains from 10 8  to 10 12  polymer molecules per vector. 
     
     
         8 . The nanoparticle of  claim 7 , wherein the nanoparticle contains from about 10 9  to about 10 10  polymer molecules per vector. 
     
     
         9 . The nanoparticle of any of the preceding claims, wherein the viral vector is a retroviral vector. 
     
     
         10 . The nanoparticle of  claim 9 , wherein the retroviral vector is a lentiviral vector. 
     
     
         11 . The nanoparticle of any of the preceding claims, further comprising one or more targeting moieties linked to one or more polymer molecules. 
     
     
         12 . The nanoparticle of  claim 11 , wherein the targeting moiety is selected from the group consisting of antibodies, antibody fragments, scFvs, antibody-like protein scaffolds, oligopeptides, aptamers, L-RNA aptamers, and ligands for cell surface receptors. 
     
     
         13 . The nanoparticle of  claim 12 , wherein the targeting moiety is an anti-CD3 antibody or an anti-CD3 aptamer. 
     
     
         14 . The nanoparticle of any of the preceding claims, wherein the transgene encodes a chimeric antigen receptor. 
     
     
         15 . The nanoparticle of any of the preceding claims that has a zeta potential from about −15 mV to about +15 mV. 
     
     
         16 . The nanoparticle of any of the preceding claims that lacks VSV-G envelope protein. 
     
     
         17 . The nanoparticle of  claim 16  that has an improved safety profile for in vivo use compared to a similar nanoparticle comprising VSV-G envelope protein. 
     
     
         18 . The nanoparticle of any of the preceding claims, wherein the viral vector lacks viral envelope protein, and wherein the nanoparticle is only capable of transducing a mammalian cell above a threshold number of polymer molecules per vector. 
     
     
         19 . The nanoparticle of  claim 18 , wherein below said threshold number of polymer molecules per vector, the amount of polymer is insufficient to completely coat the vector. 
     
     
         20 . The nanoparticle of  claim 18  or  claim 19 , wherein below said threshold number of polymer molecules per vector, the nanoparticle is structurally unstable or subject to dissociation of polymer molecules from the nanoparticle. 
     
     
         21 . The nanoparticle of any of  claims 18 - 20  that has an improved safety profile for in vivo use compared to a vector or polymer-encapsulated vector that lacks said threshold. 
     
     
         22 . A method of making the nanoparticle of any of the preceding claims, the method comprising the steps of:
 (a) providing a viral vector comprising a transgene;   (b) providing a polymer or a mixture of polymers, and optionally one or more targeting moieties; and   (c) contacting the viral vector and the polymer or the mixture of polymers, and optionally the one or more targeting moieties, whereby the viral vector and the polymer or the mixture of polymers combine to form said nanoparticle.   
     
     
         23 . The method of  claim 22 , wherein the viral vector lacks envelope protein. 
     
     
         24 . The method of  claim 22  or  claim 23 , wherein step (c) is performed at a pH of about 5.0 to about 6.5. 
     
     
         25 . The method of  claim 24 , wherein step (c) is performed at a pH of about 5.5 to 6.0. 
     
     
         26 . The method of any of  claims 22 - 25 , wherein the polymer or the mixture of polymers comprises a poly(beta-amino ester) having the formula 
       
         
           
           
               
               
           
         
         wherein each Pep is an oligopeptide; wherein R is OH, CH 3 , or cholesterol; and wherein m ranges from 1 to 20, n ranges from 1 to 100, and o ranges from 1 to 10. 
       
     
     
         27 . The method of  claim 26 , wherein each oligopeptide comprises at least three amino acid residues selected from the group consisting of R, E, K, D, H, and C. 
     
     
         28 . The method of  claim 26 , wherein the amino acid sequence of at least one of the oligopeptides is CRRR (SEQ ID NO:1), or CHHH (SEQ ID NO:2), or CKKK (SEQ ID NO:3), or CEEE (SEQ ID NO:4), or CDDD (SEQ ID NO:5). 
     
     
         29 . The method of any of  claims 22 - 28 , wherein the polymer or mixture of polymers has a net positive charge or a net negative charge at pH 7. 
     
     
         30 . The method of of any of  claims 22 - 29 , wherein the nanoparticle contains from 10 8  to 10 12  polymer molecules. 
     
     
         31 . The method of  claim 30 , wherein the nanoparticle contains from about 10 9  to about 10 10  polymer molecules. 
     
     
         32 . The method of of any of  claims 22 - 31 , wherein the retroviral vector is a retroviral vector. 
     
     
         33 . The method of  claim 32 , wherein the retroviral vector is a lentiviral vector. 
     
     
         34 . The method of of any of  claims 22 - 33 , further comprising contacting the vector with one or more targeting moieties, optionally wherein the targeting moieties are linked to the polymer or mixture of particles. 
     
     
         35 . The method of  claim 34 , wherein the targeting moiety is selected from the group consisting of antibodies, antibody fragments, scFvs, antibody-like protein scaffolds, oligopeptides, aptamers, L-RNA aptamers, and ligands for cell surface receptors. 
     
     
         36 . The method of  claim 35 , wherein the targeting moiety is an anti-CD3 antibody or an anti-CD3 aptamer. 
     
     
         37 . The method of any of  claims 22 - 36 , wherein the transgene encodes a chimeric antigen receptor. 
     
     
         38 . The method of any of  claims 22 - 37 , wherein the viral vector lacks VSV-G envelope protein. 
     
     
         39 . The method of any of  claims 22 - 38 , wherein step (c) is performed at a pH where the polymer or the mixture of polymers has a net positive charge and the retroviral vector has a negative surface potential. 
     
     
         40 . A method of treating a disease, the method comprising administering a composition comprising a plurality of nanoparticles of any of  claims 1 - 21  to a subject in need thereof, whereby cells of the subject are transduced by the retroviral vector and the transgene is expressed in the transduced cells. 
     
     
         41 . The method of  claim 40 , wherein the disease is cancer. 
     
     
         42 . The method of  claim 40  or  claim 41 , wherein the subject is human. 
     
     
         43 . A method of transducing cells in vitro, the method comprising contacting cultured cells with a plurality of nanoparticles of any of  claims 1 - 21 , whereby at least some of the cultured cells are transduced by the retroviral vector and the transgene is specifically expressed in the transduced cells. 
     
     
         44 . The method of  claim 43 , wherein the nanoparticles are targeted to CD3-positive cells and the transgene is specifically expressed in CD3-positive cells. 
     
     
         45 . The method of  claim 43  or  claim 44 , wherein the transgene encodes a chimeric antigen receptor. 
     
     
         46 . A method of performing gene therapy, the method comprising contacting cells in vitro or within a living organism with a plurality of nanoparticles of any of  claims 1 - 21 , whereby the transgene is expressed in the cells. 
     
     
         47 . A nanoparticle comprising a viral vector coated with a polymer or a mixture of polymers, wherein the viral vector comprises a reduced level of envelope protein, and comprises a transgene, wherein the nanoparticle functions as a vehicle for delivering the transgene to eukaryotic cells, and wherein the reduced level of envelope protein is insufficient to promote cellular entry of the vector and transduction of the cells by the vector. 
     
     
         48 . The nanoparticle of  claim 47 , wherein the reduced level of envelope protein is 5% or less of an amount of envelope protein in a functional virus from which the vector was derived.